Heparins are a heterogeneous group of mucopolysaccharides consisting of alternating residues of D-glucosamine and D-glucuronic acid joined by 1-4 linkages. Heparins can be found in various mammalian tissues, especially in the liver, the lung, as well as in mast cells. In the plasma, heparin is known to inhibit coagulation and also to accelerate the removal of triglycerides from the blood.
Heparins are commonly used anticoagulant drugs. The best understood action of heparin is in the inactivation of thrombin, which is thus prevented from acting on fibrinogen. Blood contains a naturally occurring inhibitor of thrombin, termed antithrombin III, which combines with the thrombin molecule and inactivates it. Heparin binds to antithrombin III, thereby markedly accelerating the reaction between thrombin and antithrombin III, and thus causing the inactivation of thrombin.
Heparin also acts earlier in the coagulation sequence by accelerating the combination of antithrombin III with other blood coagulation factors, in particular factors IXa, Xa, XIa and XIIa. Consequently, the results of all coagulation tests will be affected if heparin is present in the plasma or serum tested.
Specific methods for measuring heparin concentrations in plasma and other biological fluids are needed both to evaluate the relation between heparin concentration and its biological effects and for pharmacological studies.
Several types of heparin assays are currently available. Chemical methods, such as the uronic acid carbazole reaction, can be used to detect many different heparins but are relatively insensitive and frequently require a purified sample in order to yield quantitative results. Jacques, et al. (Analyt. Biochem. 52: 219-233, 1973) disclosed an assay for the identification and quantitation of heparins which comprised microelectrophoresis of samples on agarose gels coupled with staining with toluidine blue solutions. A linear relationship was observed between absorbance and heparin concentration applied so that the total optical density of spots could be used to estimate the heparin concentration. Although this assay permitted the measurement of heparin in complex mixtures, it was too insensitive and too cumbersome for routine use.
Biological assays for heparin are based on the overall anticoagulant activity of heparin, or on more specific properties such as the inactivation of thrombin or coagulation factors. Yin, et al., (J. Lab. Clin. Med. 81: 298-310, 1973) described a quantitative assay for heparin which was said to detect as little as 0.01 units of heparin activity (corresponding to 0.1 micrograms of heparin). The assay was based upon the accelerating effect of heparin on the neutralization of activated factor X by its plasma inhibitor, antithrombin III.
Teien, A. N. and Lie, M. (Thromb. Res. 7: 777-788, 1975) evaluated five clotting methods for the determination of heparin activity in plasma including the activated partial thromboplastin time (APTT); the calcium thrombin time; the method of Yin, et al., above; titration with Polybrene; and the method of Denson and Bonnar (Thromb. Diath. Haemorrh., 30: 4711, 1973), the latter being a modification of Yin, et al. (op. cit.).
Teien, et al. reported considerable variation between the values obtained with the different biological assays. This is not surprising because each of these biological assays depends upon a different property of the heparin molecule. The clotting assays investigated by Teien, et al. are global tests in that they measure the resultant activity arising from a balance between activators and inhibitors of the coagulation system. These assays are useful in monitoring the biological activities of the heparin molecule. However, heparin treatment itself reduces the level of antithrombin III. Moreover, in the course of a disease, the heparin sensitivity in the blood may change. In addition, heparin antagonists in the blood, such as platelet factor 4, may increase. Hence, for the objective determination of total heparin levels (independent of biological function), these assays are of limited usefulness.
In an attempt to overcome the problems inherent in biological assays such as the one described above, Dawes and Pepper (Thromb. Res. 27: 387-396, 1982), described a competitive binding assay for exogenous and endogenous heparins. In this assay, heparin is said to compete with radioactively labeled heparin for binding to protamine-Sepharose. Although this assay is said to be quite sensitive (the article reports detection of heparin at concentrations as low as 10 ng per ml), it requires an incubation period of at least 16 hours just for binding. Moreover, time is required to predigest the biological fluid to be tested in order to remove interfering substances and yield an assayable sample. The long duration of this assay and the need for predigestion make it unsuitable for clinical use and cumbersome for use in determining heparin levels in nonclinical applications. Furthermore, this assay is not specific because it also recognizes a variety of other sulfated polysaccharides, such as heparan sulfate.
Bessos, H. (Thromb. Res. 35: 267-278, 1984) described a process for degrading heparin and heparan sulfate with various chemical methods, and assaying the degradation products with the competitive binding assay of Dawes and Pepper. Treatment with nitrous acid was found to degrade heparin faster and to a greater extent than heparan sulfate. Assay of the degradation products could be used to correct the last of specificity of the competitive assay of Dawes and Pepper. However, this modification of the Dawes and Pepper assay renders the assay procedure even longer and more complex, and thus unsuitable for routine laboratory use.
Therefore, there is a need in the art to provide a system to detect heparin which is faster, more specific, sensitive and independent of any biological functions of the molecule. The utility of such an assay is manifold. For example, the assay could be used together with activity assays to determine the amount of heparin required to obtain a given antithrombotic level, thereby providing the clinician with a more precise estimate of the risk of raising or lowering the dosage of heparin employed. It also could be used as a biological probe for the presence of heparin in various cells and tissues and in solid matrices such as endothelial or heparinized synthetic surfaces, topics of increasing importance to the fields of atherosclerosis and non-thrombogenic surfaces, including artificial hearts and heart valves. Finally, there is a need for an assay that distinguishes between heparin and related glycosaminoglycans.
The present inventors have devised a rapid, sensitive and specific assay for heparin in biological fluids. As used in this application, biological fluids include but are not limited to plasma, urine, cell suspensions, tissue extracts and other bodily and physiological fluids which may contain heparin. This assay and the reagents employed herein are also useful during the commercial scale isolation and purification of heparin for pharmacological use, and as a probe for the presence of heparin in tissues.